FKBP6 Human

What is FKBP6 and what are its structural characteristics?

FKBP6 (FK506 binding protein 6) is a human gene located on chromosome 7q11.23 that encodes a protein structurally similar to FKBP immunophilins, which are known to bind to immunosuppressants FK506 and rapamycin . The protein has a distinct structural composition comprising 3 α-helices and 11 β-sheet strands, characteristic of the FK506-binding protein family . Unlike other family members, FKBP6 contains a peptidyl-prolyl isomerase (PPIase) domain that has lost its isomerase activity, which typically catalyzes the transformation of proline residues from trans to cis form .

The protein structure also features a tetratricopeptide repeat (TPR) domain in its downstream region, which facilitates critical protein-protein interactions, particularly with heat shock proteins . This domain architecture is fundamental to FKBP6's biological functions, especially its role in spermatogenesis and the piRNA pathway. The identification of several key protein-protein interaction interfaces has informed structure-based studies, revealing how FKBP6 engages with components of the piRNA biogenesis machinery and meiotic structures.

What is the expression pattern and tissue specificity of FKBP6?

FKBP6 demonstrates highly tissue-specific expression, with studies confirming that the human FKBP6 gene is primarily expressed in the testis . This restrictive expression pattern aligns with its crucial role in male fertility and spermatogenesis. Within testicular tissue, FKBP6 protein localizes to meiotic chromosome cores and regions of homologous chromosome synapsis, highlighting its fundamental role in spermatocyte development .

The testis-specific expression can be quantified using RT-qPCR and visualized through immunofluorescence staining, techniques that have been employed to confirm lack of FKBP6 expression in testicular tissue of individuals with homozygous loss-of-function variants . When examining the expression across different pathological states, it's noteworthy that FKBP6 mRNA expression levels showed no significant differences between lung cancer tissues and healthy samples, unlike some other members of the FKBP family that display altered expression in malignancies .

How does FKBP6 differ functionally from other FKBP family members?

FKBP6 diverges functionally from other FKBP family members in several significant aspects. While many FKBPs retain active peptidyl-prolyl isomerase activity that catalyzes proline isomerization, FKBP6 has evolutionarily lost this enzymatic capability . This functional divergence suggests that FKBP6 has evolved specialized roles distinct from the canonical functions of the FKBP family.

How does FKBP6 contribute to male fertility and spermatogenesis?

FKBP6 plays a crucial role in male fertility through its essential functions during spermatogenesis. Research has established that FKBP6 is indispensable for homologous chromosome pairing during meiosis in spermatogenesis . In experimental models, targeted inactivation of FKBP6 in mice results in infertile males while females remain apparently normal, underscoring its sex-specific reproductive function .

At the molecular level, FKBP6 facilitates two critical processes during spermatogenesis. First, it contributes to meiotic progression through its association with chromosomal structures, though interestingly, human FKBP6 was not detected as part of the synaptonemal complex in normal human spermatocytes, contrary to findings in mice . Second, FKBP6 has a vital role in piRNA biogenesis, with small RNA sequencing revealing that loss of FKBP6 severely impacts piRNA levels in human testicular tissue . This dual functionality explains why FKBP6 deficiency leads to spermatogenic arrest at the round spermatid stage in humans, resulting in azoospermia or extreme oligozoospermia that is not amenable to assisted reproductive technologies .

What methodologies are used to study FKBP6 mutations in human male infertility?

The investigation of FKBP6 mutations in the context of human male infertility employs several sophisticated methodological approaches. Exome sequencing represents the primary tool for identifying pathogenic variants in FKBP6 among infertile men . This approach has been successfully implemented in multiple research cohorts including the MERGE cohort (1,671 men with various infertility phenotypes), the GEMINI cohort (1,011 men with spermatogenic failure), and the Imperial cohort (17 azoospermic men) .

Following variant identification, functional validation of FKBP6 mutations involves multiple techniques. RT-qPCR is employed to quantify FKBP6 expression levels in testicular tissue, while immunofluorescence staining allows visualization of protein localization patterns . For deeper molecular insights, small RNA sequencing is utilized to assess the impact of FKBP6 variants on piRNA biogenesis and abundance .

Histological analysis of testicular tissue through testicular sperm extraction (TESE) or testicular biopsy provides critical morphological information about spermatogenic arrest patterns in men with FKBP6 mutations . These combined approaches have established that FKBP6 deficiency typically leads to arrest at the round spermatid stage, a distinctive phenotype that guides clinical diagnosis and management .

What is the genetic evidence linking FKBP6 variants to azoospermia?

The genetic evidence connecting FKBP6 variants to azoospermia has been substantiated through multiple independent studies. A mutation analysis covering all coding regions of the human FKBP6 gene in 19 patients with azoospermia resulting from meiotic arrest identified a novel polymorphism site, 245C → G (Y60X) in exon 3 . This finding, along with the analysis of two polymorphism sites, suggested that the human FKBP6 gene might be imprinted in the testis .

More definitive evidence emerged from the identification of homozygous loss-of-function variants in six individuals who presented with no or extremely few sperm in the ejaculate, rendering them unsuitable for medically assisted reproduction . Evaluation of testicular tissue from these individuals consistently revealed an arrest at the stage of round spermatids . The causal relationship was further confirmed by the absence of FKBP6 expression in testicular tissue as demonstrated by RT-qPCR and immunofluorescence staining .

The genetic architecture of FKBP6-associated azoospermia has been characterized through screening of multiple cohorts totaling over 2,600 men with infertility, which has enhanced our understanding of the prevalence and spectrum of pathogenic variants in this gene . These findings collectively establish FKBP6 as a clinically significant gene for genetic diagnosis of male infertility.

What is known about FKBP6's role in the piRNA pathway and its relation to infertility?

FKBP6's critical involvement in the piRNA pathway represents a key mechanism underlying its essential role in spermatogenesis. In humans, loss of FKBP6 severely impacts piRNA levels, confirming its function in piRNA biogenesis . This finding is consistent with studies in mice where Fkbp6 directly interacts with central piRNA components including Miwi (Piwil1), Miwi2 (Piwil4), and Tdrd1, which are known to be essential for piRNA biogenesis .

The molecular interaction network of FKBP6 in the piRNA pathway is facilitated by its tetratricopeptide repeat (TPR) domain, which mediates protein-protein interactions, particularly with heat shock proteins . In mice, Fkbp6 has been shown to bind the heat shock protein Hsp90, which is involved in the piRNA pathway . This interaction appears to be conserved and functionally significant for piRNA biogenesis across species.

Interestingly, despite FKBP6's role in the piRNA pathway, loss of FKBP6 in humans does not lead to increased LINE-1 expression, which contrasts with findings in other piRNA-pathway mouse models . This discrepancy suggests species-specific differences in how piRNA pathway components regulate transposable elements or indicates that FKBP6 may have a more specialized role within the piRNA biogenesis machinery in humans compared to mice.

How do researchers investigate protein-protein interactions involving FKBP6?

Investigating protein-protein interactions (PPIs) involving FKBP6 requires multiple complementary methodologies. Researchers employ yeast two-hybrid (Y2H) screening as an initial approach to identify potential interaction partners, as demonstrated in studies examining FKBP family member interactions . This technique allows for the systematic identification of proteins that physically associate with FKBP6 in a cellular context.

Computational approaches complement experimental methods, with tools like GeneMANIA and STRING databases being utilized to predict and visualize PPIs involving FKBP members including FKBP6 . These analyses have revealed that FKBP family members, including FKBP6, interact with genes like HECTD1 and TTC6 through shared protein domains (55.30%), coexpression (31.22%), physical interactions (13.16%), and other mechanisms .

For targeted validation of specific interactions, co-immunoprecipitation followed by mass spectrometry provides more definitive evidence of physiologically relevant protein complexes containing FKBP6. These techniques have helped establish FKBP6's interactions with heat shock proteins like Hsp90 and with piRNA pathway components such as Piwil1, Piwil4, and Tdrd1 . The functional significance of these interactions can be further assessed through in vitro binding assays measuring interaction affinity and specificity, and through in vivo studies examining the consequences of disrupting specific interaction interfaces.

What experimental strategies are used to model FKBP6 deficiency?

Modeling FKBP6 deficiency to understand its functional implications employs diverse experimental strategies across different biological systems. Mouse models have been instrumental, with targeted inactivation of Fkbp6 in mice resulting in infertile males but apparently normal females, establishing the sex-specific reproductive consequences of FKBP6 deficiency . These mouse models have allowed detailed characterization of the spermatogenic defects, revealing abnormal pairing and misalignment of homologous chromosomes, non-homologous partner switches, and autosynapsis of the X chromosome cores in meiotic spermatocytes .

CRISPR/Cas9-mediated genome editing provides a powerful approach for evaluating potential human disease variants in FKBP6. This methodology allows researchers to model specific missense variants identified in human populations to assess their functional consequences in an appropriate in vivo system . The selection of candidate infertility missense variants for modeling typically follows strict criteria: they reside in genes essential for fertility in mice, alter an amino acid conserved between mice and humans, and are predicted to be deleterious by various bioinformatic tools .

Human studies utilizing testicular biopsies from individuals with naturally occurring FKBP6 variants offer the most clinically relevant insights. These studies employ a comprehensive toolkit including RT-qPCR, immunofluorescence staining, and small RNA sequencing to characterize the molecular and cellular consequences of FKBP6 deficiency in the authentic human context . This multi-level approach allows researchers to bridge findings from model organisms to human pathophysiology, establishing FKBP6's essential role in human male fertility.

How does FKBP6 relate to Williams syndrome and other genetic disorders?

FKBP6 is located on chromosome 7q11.23, a region deleted in Williams syndrome, making FKBP6 one of the genes that experiences hemizygous loss in this multisystem developmental disorder . Interestingly, despite this hemizygous deletion of FKBP6 in Williams syndrome patients, this partial loss is not associated with infertility . This observation suggests that a single functional copy of FKBP6 is sufficient to maintain normal spermatogenesis, aligning with the recessive inheritance pattern of FKBP6-related infertility.

The relationship between FKBP6 and Williams syndrome highlights the importance of gene dosage in understanding phenotypic manifestations. While complete loss of FKBP6 function leads to male infertility, the hemizygous state appears compatible with normal reproductive function. This differential impact provides valuable insights into the threshold requirements for FKBP6 in various biological contexts and exemplifies how genomic disorders affecting multiple genes can help delineate dosage-sensitive versus dosage-insensitive functions.

This clinical correlation also informs research methodology by demonstrating the value of examining natural genetic variants across different populations. By studying individuals with distinct genetic alterations affecting FKBP6—from hemizygous deletions in Williams syndrome to homozygous loss-of-function variants in infertility cases—researchers can develop more nuanced models of gene function that account for dosage effects and genetic background influences.

What is the prevalence and spectrum of pathogenic FKBP6 variants in infertile populations?

The prevalence and spectrum of pathogenic FKBP6 variants among infertile men have been investigated through comprehensive genetic screening of multiple cohorts. Studies have examined large cohorts including the MERGE cohort (1,671 men with various infertility phenotypes), the GEMINI cohort (1,011 men with spermatogenic failure), and the Imperial cohort (17 azoospermic men), identifying individuals with bi-allelic pathogenic variants in FKBP6 .

A novel polymorphism site, 245C → G (Y60X) in exon 3, was identified in studies involving 19 patients with azoospermia resulting from meiotic arrest . Further research has expanded the catalog of pathogenic variants, establishing a clearer picture of the genetic architecture of FKBP6-associated infertility. The consistent phenotypic presentation—azoospermia or extreme oligozoospermia with arrest at the round spermatid stage—suggests that complete loss of FKBP6 function has predictable reproductive consequences .

The identification of homozygous loss-of-function variants in six individuals from independent studies confirms that FKBP6-related infertility follows an autosomal recessive inheritance pattern . This genetic information is valuable for clinical genetic counseling and in guiding expectations about the likelihood of successful sperm retrieval through testicular sperm extraction procedures. The expanding catalog of FKBP6 variants also enhances our ability to offer precise genetic diagnosis to infertile men, particularly those with non-obstructive azoospermia.

What potential role does FKBP6 play in cancer biology?

The relationship between FKBP6 and cancer biology represents an emerging area of investigation with contrasting findings across different malignancies. In lung adenocarcinoma (LUAD), mRNA expression levels of FKBP6 showed no significant differences between lung cancer tissues and healthy samples, distinguishing it from several other FKBP family members that display altered expression patterns in this cancer type . This suggests that FKBP6 may not contribute significantly to LUAD pathogenesis, unlike some of its family members.

The methylation status of FKBP6 has been identified as a potential biomarker in cervical cancer, suggesting that epigenetic regulation of this gene might play a role in certain malignancies . This finding highlights the importance of considering multiple regulatory mechanisms when investigating FKBP6 in cancer biology, including not only expression levels but also epigenetic modifications, post-translational modifications, and alterations in protein-protein interactions that might affect its function in the cancer context.

How do researchers evaluate potentially pathogenic FKBP6 variants?

The evaluation of potentially pathogenic FKBP6 variants employs a multi-tiered approach combining computational prediction, in vitro functional assessment, and in vivo modeling. Initially, variants are selected based on several criteria: they reside in genes essential for fertility in mice, alter amino acids conserved between mice and humans, and are predicted to be deleterious by various bioinformatic tools such as SIFT and PolyPhen2 .

To increase the success rate in identifying truly pathogenic missense variants, researchers have implemented additional in vitro prescreening steps. One approach prioritizes variants that disrupt known protein-protein interactions, as human disease-causing Mendelian alleles are overrepresented among variants affecting interaction interfaces . A second screening identifies variants that destabilize the protein in cultured cells, as protein stability often correlates with functional integrity .

For definitive functional assessment, CRISPR/Cas9-mediated genome editing allows researchers to model specific variants in mice, providing an in vivo system to evaluate their effects on fertility and spermatogenesis . This integrated computational and experimental approach has proven valuable for functionally assessing potentially deleterious missense variants in essential reproductive genes, including FKBP6. The combination of these methodologies enhances our ability to distinguish pathogenic variants from benign polymorphisms, which is crucial for accurate genetic diagnosis and counseling in the clinical setting.

What are the key outstanding questions in FKBP6 research?

Despite significant advances in understanding FKBP6's role in spermatogenesis and male fertility, several key questions remain unanswered. A fundamental outstanding question concerns the precise molecular mechanism by which FKBP6 contributes to piRNA biogenesis in humans. While its involvement in the piRNA pathway has been established, the exact steps of the pathway that require FKBP6 and the molecular details of its contribution remain to be fully elucidated .

Another intriguing question arises from the species-specific differences observed in FKBP6 function. In mice, Fkbp6 has been identified as a component of the synaptonemal complex, whereas in humans, FKBP6 was not detected as part of this structure in normal spermatocytes . Additionally, unlike findings in piRNA-pathway mouse models, loss of FKBP6 in humans does not lead to increased LINE-1 expression . These disparities raise questions about the evolutionary divergence of FKBP6 functions and the species-specific adaptations in the pathways it regulates.

The potential functions of FKBP6 beyond reproduction remain largely unexplored. Given its classification as an immunophilin and its structural homology to proteins that bind immunosuppressants like FK506 and rapamycin, FKBP6 might have immunological functions that have not yet been characterized . Similarly, its potential roles in other cellular processes, perhaps in specific contexts or cell types beyond the testis, represent areas for future investigation. These outstanding questions highlight the need for continued research on this multifaceted protein to fully understand its biological significance.